This invention relates to analytical tools and methods for monitoring levels of gene expression and mutations in gene sequences. In particular, the invention relates to assay systems, tools and methods with enhanced specificity and sensitivity that are capable of multiplexing one or more sample(s), having one or more target material(s) per sample, on a single array.
Conventional analysis of biological materials, such as DNA, RNA, proteins, antibodies, ligands and the like, employs a basic hybridization assay which comprises a substrate or support having biological material chemically bound thereto. The biological material may be either biological xe2x80x9cprobesxe2x80x9d of known molecular make-up or xe2x80x9ctargetsxe2x80x9d having an unknown characteristic to be determined. For the purposes of simplicity, hereinafter the material bound to the substrate will be referred to as probes.
The probes are hybridized with a target sample and the hybridization results are analyzed. The hybridization results reveal information about the targets based on what is known about the probes. The surface bound probes are typically formed of DNA oligonucleotides, cDNA""s, PCR products, proteins, antibodies, antigens, receptors, ligands, and the like, that are complementary to the biological target material under test.
Another conventional assay is the sandwich hybridization assay. Sandwich hybridization assays use probes designed with a sequence region that is complementary to the target under test and a separate sequence region or a separate binding partner that is complementary to a sequence, or specific to a binding partner, on a support. The probes are hybridized with the target sample and with its complement on the support in a two step process. Variations on this basic scheme have been developed to enhance accuracy, facilitate separation of duplexes and amplify signals for detection during analysis (see for example, U.S. Pat. Nos. 4,868,105; 5,200,314; 5,635,352; and 5,681,697, issued to Urdea (or Urdea et al.) and U.S. Pat. Nos. 5,681,702 and 5,780,610, both issued to Collins et al.).
However, a drawback to the sandwich hybridization technique is cross hybridization. For example, if the target material hybridizes to the wrong region of the probe, then the probe does not hybridize with its appropriate complement on the support. This may yield a false negative result. Conversely, if the target material hybridizes incorrectly to the sequence on the support, a false positive result may occur. Thus, information about the target material becomes less accurate. There has been much effort in developing methods for minimizing cross-hybridization in sandwich hybridization assays. U.S. Pat. No. 5,604,097, U.S. Pat. No. 5,635,400 and U.S. Pat. No. 5,846,719, issued to S. Brenner and Brenner et al., respectively (hereinafter xe2x80x9cBrennerxe2x80x9d), disclose methods of sorting polynucleotides in basic hybridization assays usingxe2x80x98minimally cross-hybridizationxe2x80x99 sets of oligonucleotide tags. Brenner is silent on using the methods of sorting for sandwich hybridization assays. Oligonucleotide tags from the set of tags are attached to a sample of polynucleotides under test. The polynucleotides with oligonucleotide tags attached are immobilized on a solid phase support by hybridizing the tags to a complementary sequence on the support.
Brenner discloses a general algorithm and computer program for computing minimally cross-hybridizing sets of tags and complements. Brenner""s test for xe2x80x9cminimally cross-hybridizingxe2x80x9d is based upon the conventional technique of symbolic matching of sequences. Although useful in some cases, the conventional symbolic matching technique has drawbacks that affect the technique""s ability to effectively discriminate against cross-hybridizations. Since some base mismatches are much less destabilizing to the duplex Tm than other mismatches, the method of Brenner is capable of generating mismatch sequences which are actually capable of cross-hybridizing. The conventional method and the method disclosed by Brenner do not take in account cross-hybridizing mismatches. In addition, Brenner does not teach a method for protecting against the formation of intramolecular structures. These structures, such as hairpins, will inhibit the correct duplex formation between tags and their complements. If cross-hybridizing mismatches and intramolecular structures were screenable according to Brenner""s method, the number of tags and complement sets after such screening, which would actually qualify as xe2x80x9cminimally cross-hybridizingxe2x80x9d, would be greatly reduced. With state-of-the-art arrays containing more than 10,000 features, the tag sets disclosed by Brenner would have to have longer lengths than that disclosed by Brenner in order to yield a high enough number to accommodate such an array. However, longer length tags and complements are more expensive to synthesize.
Thus, it would be advantageous to have a large number of xe2x80x98tag and complementxe2x80x99 sets, for example, for use in diagnostic assays of biological materials, wherein the tag and complement lengths are as short as possible to save on cost. Further, it would be advantageous if the specificity between the tags and their complements was increased to avoid or minimize cross-hybridizations and still further if the sensitivity between the tags and their complements was increased by decreasing the probability of intramolecular structures, such as hairpins, within the sequences. Still further, it would be advantageous if such tag and complement sets could be adapted to sandwich hybridization assays using arrays of over 10,000 features.
U.S. Pat. No. 5,399 676, U.S. Pat. No. 5,527,899, and U.S. Pat. No. 5,721,218 issued to B. Froehler, disclose using oligonucleotides with xe2x80x9cinverted polarityxe2x80x9d for forming anti-sense probes having an extended triple helix with a double-helical nucleotide duplex. The anti-sense probes are used in clinical intervention applications to decrease specific RNA translation. Froehler discloses that the inverted polarity oligonucleotides can skip from one complementary strand in the duplex to the other as its polarity shifts. Such inverted polarity also stabilizes the single-stranded oligonucleotides to exonuclease degradation. However, Froehler is silent on using inverted polarity oligonucleotides for minimizing cross hybridization in diagnostic assays. In addition, Froehler discloses using probes that actually have specific intramolecular structures, which is consistent with the use of anti-sense probes in clinical intervention applications.
Thus, it would be advantageous to have tools and methods for diagnostically assaying one or more biological sample(s), having one or more target(s) per sample, on a single array, using sandwich hybridization assay techniques. In addition, it would be advantageous for the tools and methods to have increased specificity between complementary probe sequences to minimize the likelihood of cross-hybridization between biological materials in a systematic fashion. Moreover, it would be advantageous for such tools and methods to have increased sensitivity between complementary probe sequences to minimize the likelihood of intramolecular structures within the probes. The increased specificity and sensitivity of such tools and methods would increase the accuracy and usefulness of sandwich hybridization assays, especially on an array.
The present invention provides sandwich hybridization assay systems, biological tools and methods of diagnostically assaying biological materials. The present invention is particularly useful for sandwich hybridization assays on arrays and provides an addressable, self-assembling array. The systems, tools and methods are capable of multiplexing one or more sample(s), having one or more target(s) per sample on a single array. Moreover, the systems, tools and methods of the invention have good specificity by systematically providing a reduced likelihood of cross-hybridizations from occurring, and good sensitivity by systematically providing a reduced likelihood of intramolecular structures from forming. The sandwich hybridization assay systems, tools and methods of the present invention provide powerful means for sorting, tracking, identifying, and determining other characteristics of biological target compounds for diagnostic applications.
According to one aspect of the present invention, an assay system for multiplexing on a single array one or more biological sample(s), having one or more biological target(s) per sample, is provided. The assay system for multiplexing comprises an array apparatus that has a first plurality of biological probes, called capture probes, in an array pattern of features on a substrate. Each capture probe in each feature location is different from the others in the first plurality. Each different capture probe is a different address on the array apparatus.
The assay system for multiplexing still further comprises a second plurality of biological probes, called solution probes. Each solution probe comprises a first region and a second region. Each solution probe is different from others in the second plurality by comprising a different first region, and may comprise a different second region, depending on the assay. The first region of each solution probe is complementary to a respective capture probe on the array. The second region of the solution probe is complementary to a respective biological target in a sample. Thus, the solution probes essentially assemble or deliver different biological target-and-biological sample combinations being assayed on the array corresponding to the addresses of the different first probes. The presence, quantity and/or other features of particular targets in particular samples are ascertainable depending on whether the particular target-sample combinations have been assembled to their respective capture probe location on the array during the assay.
According to another aspect of the present invention, an assay method of multiplexing on a single array one or more biological sample(s), having one or more biological target(s) per sample, is provided. The assay method of multiplexing comprises the step of providing an array apparatus that has a first plurality of biological probes, called capture probes, in an array pattern of features on a substrate. Each capture probe of the first plurality is different and each different capture probe is located in a different feature location of the array. Each different capture probe is an address on the array apparatus.
The assay method of multiplexing further comprises the step of providing a second plurality of biological probes, called solution probes. Each solution probe of the second plurality has a first region and a second region. Each solution probe is different from other of the second plurality by having a different first region. Each different first region is complementary to a different capture probe on the array. The second region is complementary to a biological target from a biological sample, and therefore, the second region may be the same or different on each solution probe of the second plurality, depending on the assay to be performed. As mentioned above for the system, the solution probes essentially assemble or deliver the different biological target-and-biological sample combinations being assayed on the array corresponding to the addresses of the different capture probes. The assay method of multiplexing still further comprises the steps of assembling the biological target(s) from the sample(s) to the array, and removing unassembled biological materials from the array and analyzing the assay results using conventional methods. The presence of the biological target(s) from respective biological sample(s) at corresponding capture probe feature locations on the array indicates, among other things, whether and how much of particular biological targets exist in particular biological samples.
During the step of assembling, a target from a sample will hybridize with a complementary second region of a solution probe and the first region of this solution probe will hybridize with a complementary capture probe on the array corresponding to the target-sample combination. The solution probes of the system essentially assemble or xe2x80x9caddressxe2x80x9d the targets to the array in a predetermined (pre-addressed) fashion during the assay. The assay system and method for multiplexing of the present invention advantageously provide an assay that is self-assembling and addressable, and capable of sorting for evaluation purposes: one target from a plurality of different samples (or patients), a plurality of different targets from one sample (patient), or a plurality of different targets from a plurality of different samples on the same array. The hybridizations may be performed simultaneous or preferably, in a two step hybridization process.
According to still another aspect of the present invention, a set of biological probes used in a sandwich hybridization assay of a biological target on an array of biological features is provided. The set of biological probes comprises a plurality of individual solution probes that each comprises a first probe region, called an anti-capture region, complementary to a biological feature on the array. Each individual solution probe further comprises a second probe region that is complementary to the biological target being assayed. Each solution probe in a particular set is different by comprising a different anti-capture region. The anti-target region on each different solution probe of a set may be the same or different depending on the type of assay being performed. Moreover, there may be multiple copies of each different solution probe in the set. There is a different set of biological probes for each type of biological material being assayed (e.g., nucleic acids, proteins, sugars, etc.).
Thus, each solution probe of a particular set may comprise first regions selected from oligonucleotides, antibodies, antigens, ligands and receptors, for example, depending on the biological make-up of the biological features on the array. Further, the second regions of this particular set may comprise second regions selected from cDNA, PCR products, oligonucleotides, antibodies, antigens, ligands and receptors, for example, depending on the biological make-up of the biological targets to be assayed. For example, each solution probe of a particular set may comprise a different antigen linked to the same or a different cDNA, wherein the different antigens are complementary to a plurality of different antibody features on an array and the cDNA is complementary to oligonucleotide (e.g., mRNA) target material(s) to be assayed. Thus, each set of solution probes is customized to a particular assay. The generic set of capture probes on the universal array apparatus provides xe2x80x9caddressesxe2x80x9d corresponding to the location of the capture probes on the array where target material being assayed is to be delivered and the set of solution probes essentially delivers the target material to its respective address during the assay. The set of biological probes are particularly useful for multiplexing assays of one or more biological sample(s), having one or more biological target(s) per sample, on a single array.
In still another aspect of the present invention, a system for assaying biological materials having specificity and sensitivity is provided. The system comprises an apparatus that has a first set of biological material probes, called capture probes, on a substrate. Each capture probe of the set comprises a sequence of monomers. Each set of capture probes is generic to the biological material to be tested. Each capture probe in the set may be different by having a different sequence of monomers. There may be multiple copies of each different capture probe in the set.
The system having specificity and sensitivity further comprises a second set of biological material probes, called solution probes. There is a different set of solution probes for each type of biological material being tested. Each solution probe of the set comprises a first sequence region, called an anti-capture sequence, that is complementary to the monomers in the capture probe sequence for hybridizing or binding to the capture probe, and a second region, called an anti-target region, for hybridizing or binding to the target. Each solution probe in a particular set may be different by comprising a different anti-capture sequence. Further, each solution probe may comprise the same or different anti-target region depending on the biological materials to be assayed. There may be multiple copies of each different solution probe in the set. The set of solution probes assembles the target material to be tested to the assay substrate by hybridizing the second region to the target and hybridizing the first region to the capture probes on the substrate. The hybridizations may be performed simultaneously or preferably, in two hybridization steps.
In accordance with this aspect of the invention, the capture probes and the anti-capture sequences on the solution probes each comprise a complementary chemically modified monomer that preferentially hybridize or bind to each other instead of to a complementary unmodified (or not similarly modified) monomer. The preference of the modified monomers of the present invention provides specificity and sensitivity to the system. The system is specific because the chemically modified monomer in the anti-capture sequences will preferentially hybridize or bind with the complementary similarly modified monomer in the respective capture probe, and likewise the modified monomer in the capture probe will preferentially hybridize or bind with the similarly modified monomer in the complementary anti-capture sequence. Thus, cross-hybridizations (i.e., hybridizations with mismatches) are less likely to occur. The system is sensitive because the chemically modified monomers in the capture probes and anti-capture sequences are less likely to hybridize to complementary unmodified (or not similarly modified) monomers or to noncomplementary monomers present in the capture probes, solution probes or target sequences. Thus, hybridizations that cause intramolecular structures within the capture probes or within the anti-capture sequences are less likely to occur.
In yet another aspect of the present invention, a method of assaying biological materials having specificity and sensitivity is provided. The method comprises the step of providing an apparatus having a first set of biological probes, called capture probes, comprising a sequence of monomers, on a substrate.
The method having specificity and sensitivity further comprises the step of providing a second set of biological probes, called solution probes. There is a different set of solution probes for each type of biological material to be tested. Each solution probe of the set comprises a first sequence region of monomers, called an anti-capture sequence region, and a second region, called an anti-target region. The anti-capture sequence region of each solution probe is complementary to the monomer sequence of a capture probe in the set, and the anti-target region on the solution probe is complementary to a biological target to be assayed.
The complementary capture probes and anti-capture monomer sequences each include a complementary chemically modified monomer that prefers to hybridize or bind to each other instead of to a complementary monomer that is not similarly modified.
The method of assaying biological materials having specificity and sensitivity further comprises the step of assembling the target material to the substrate for evaluation. During the step of assembling, the set of solution probes is incubated with a target biological material to cause hybridization or binding between targets and respective complementary anti-target regions of the solution probes. Further, the set of solution probes is incubated with the set of capture probes on the assay apparatus to cause hybridization or binding between complementary capture sequences and anti-capture sequences of the solution probes. It is during the step of assembling that the complementary chemically modified monomers on the capture probes and anti-capture sequences preferentially hybridize to each other. The hybridizations may be performed simultaneously or preferably, in two hybridization steps. After hybridization, the incubated substrate is washed to remove unhybridized/unbound material and the results of the assay are analyzed, according to conventional methods.
In a preferred embodiment, the capture probe and anti-capture sequences are oligonucleotides and the chemically modified monomers in the capture probes and anti-capture sequences are reversed polarity nucleotides relative to the polarity of the nucleotides of the respective sequence. The anti-capture sequence, having a nucleotide with reversed polarity, is complementary to the nucleotide with reversed polarity on the capture probe. The complementary reversed polarity nucleotides prefer to hybridize to each other because they form a thermodynamically more stable hybridization than a hybridization between a reversed polarity nucleotide and its complementary nucleotide whose polarity is not similarly reversed.
The chemical modification introduced into the probes of the system and method essentially improves the likelihood that the appropriate or intended capture probes and solution probes will specifically and sensitively hybridize or bind together. Advantageously, the system and method of assaying essentially systematically provide a reduced likelihood of cross-hybridizations between the capture probes and sequences that are not complementary to the capture probes, such as non-complementary anti-capture sequences of the solution probes, anti-target regions of solution probes, or target sequences. Further, the system and method having specificity and sensitivity essentially systematically provide a reduced likelihood of cross-hybridization between anti-capture sequences of the solution probes and sequences that are not complementary to the anti-capture sequences, such as non-complementary capture sequences on the array, other anti-capture sequences, anti-target sequences of solution probes, or target sequences. In addition, the system and method having specificity and sensitivity essentially systematically provide a reduced likelihood of any undesirable formation of intramolecular structures within the capture probes and anti-capture sequences of the invention. The system and method have a reduced likelihood of cross-hybridizations and intramolecular structures because the probes comprise modified monomers that prefer to hybridize to each other rather than to a complementary unmodified (or not similarly modified) monomer.
The system and method having specificity and sensitivity are useful in sandwich hybridization assays, sandwich hybridization assays using arrays, and in particular, in the system and method described above for multiplexing one or more biological sample(s), having one or more target(s) per sample on a single array. When used in multiplexing array assay applications, the system and method having specificity and sensitivity advantageously provide assays with specific and sensitive addressing and self-assembling capabilities.
The system and method having specificity and sensitivity of the invention uses both chemically modified monomers and monomers that are not modified (or not modified in a similar fashion to the chemically modified monomers of the invention), in the same capture probes and anti-capture sequences. Using both chemically modified monomers and not similarly modified monomers increases the number of different monomers (xe2x80x9clettersxe2x80x9d) available with which to make new sequences (xe2x80x9cwordsxe2x80x9d) for the capture probes and anti-capture sequences of the invention. Thus, a larger number of different capture probe sequences and their complementary anti-capture sequences are readily and systematically generated by the present invention that are specific and sensitive compared to conventional assays.
In the preferred embodiment of oligonucleotide capture probes and anti-capture sequence regions of the solution probes of the system and method having specificity and sensitivity, there are at least eight nucleotides (four with one polarity and four counterparts with respectively reversed polarity) from which to form the complementary capture probe and anti-capture region sequences. Therefore, a much larger set of probes and anti-capture sequences with specificity and sensitivity are provided. Moreover, the specificity that the complementary reversed polarity nucleotides have for each other allows the length of the capture probes and anti-capture regions to be shorter than conventional probes to save on cost.
The system and method having specificity and sensitivity advantageously provide a set of greater than 10,000 probes, which are unique with respect to each other. Mammalian genomes are estimated to contain over 10,000 expressed genes. With multiple splicing variants, the number of probes needed to sample the expressed genes is even higher. Thus, if one wanted to assay for all of the expressed genes and variants on the same array, one would need in excess of 10,000 biological capture features on the array. The system and method having specificity and sensitivity provide this capability.
In another aspect of the present invention, a kit is provided that comprises the assay apparatus of capture probes and the set of solution probes packaged with written instructions for use in accordance with one or more assay methods of the present invention. The kit can be provided to users, such as diagnostic, research and/or analytical laboratories. The kit can comprise either (i) the array apparatus of capture probes and set of solution probes for multiplexing sandwich assays on arrays, or (ii) the apparatus of capture probes and a set of solution probes for sandwich assays having specificity and sensitivity, or (iii) a combination of both, comprising the array apparatus of capture probes and the set of solution probes having specificity and sensitivity for multiplexing sandwich assays, in accordance with one or more embodiments of the invention.
Moreover, the systems, tools and methods of the present invention also provide cost effective custom assays. Customization of an assay resides in the preparation of the second set of probes (solution probes), rather than in the preparation of the first set of probes (capture probes) or the array apparatus of capture probes. Therefore, large numbers of generic or universal assay substrates with bound capture probes can be manufactured at a time, thereby saving in cost and turnaround time for custom orders. This is particular advantageous for large numbers of generic or universal array substrates of the invention. The second set of probes are prepared separately, as needed, or prepared in advance and stored separately in solution or dry, preferably frozen, until needed. The solution probes are customized to the biological materials of the generic or universal array apparatus and to the biological targets to be assayed.